Reelin-Dependent Apoer2 Downregulation Uncouples Newborn Neurons from Progenitor Cells

1258 Research Article Reelin-dependent ApoER2 downregulation uncouples newborn neurons from progenitor cells

F. Javier Pe´rez-Martıńez1,A´ lvaro Luque-Rıó2, Akira Sakakibara3, Mitsuharu Hattori4, Takaki Miyata3 and Juan M. Luque1,*,` 1Instituto de Neurociencias, Universidad Miguel Hernańdez-Consejo Superior de Investigaciones Cientı´ficas, Campus de San Juan, E-03550 San Juan de Alicante, Alicante, Spain 2Facultad de Ciencias, Universidad de Alicante, Campus de San Vicente del Raspeig, E-03690 San Vicente del Raspeig, Alicante, Spain 3Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan 4Department of Biomedical Science, Graduate School of Pharmaucetical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizzuho-ku, Nagoya, Aichi 467-8603, Japan *Present address: San Vicente 106, E-03560 El Campello, Alicante, Spain Àuthor for correspondence ([email protected])

Biology Open 1, 1258–1263 doi: 10.1242/bio.20122816 Received 11th August 2012 Accepted 17th September 2012

Summary Reelin and its receptor machinery are well known to be improves neuronal migration and positioning. Our study required for the migration and positioning of neocortical provides groundwork for the highly orchestrated clearance projection neurons. More recently, reelin has been shown both of neocortical neurons from their birth site, suggesting that necessary and sufficient to determine the rate of neocortical a reelin-dependent ApoER2 downregulation mechanism neurogenesis. The molecular links underlying its seemingly uncouples newborn neurons from progenitor cells, thereby distinct proliferative and post-proliferative functions remain enabling neurons to migrate. unknown. Here we reveal an enriched expression of functional reelin receptors, largely of Apolipoprotein E Receptor 2 2012. Published by The Company of Biologists Ltd. This is (ApoER2), in radial glia basal processes and intermediate an Open Access article distributed under the terms of the progenitor cells during mid/late cortical development. In vivo, Creative Commons Attribution Non-Commercial Share Alike

Biology Open ApoER2 overexpression inhibits neuronal migration. In License (http://creativecommons.org/licenses/by-nc-sa/3.0). contrast, precluding excessive levels of ApoER2 in reelin- deficient cortices, by either ApoER2 knock-down or the Key words: Reelin, ApoER2, Dab1, Neurogenesis, Neuronal transgenic expression of reelin in neural progenitor cells, migration, Mouse

Introduction Lipoprotein Receptor (Vldlr) induces phosphorylation of the Excitatory long distance projecting neurons originate from neural cytoplasmic adaptor protein Disabled 1 (Dab1), a tyrosine kinase progenitor cells (NPCs) locally arranged in the ventricular (VZ) signal transduction cascade, and Dab1-regulated turnover. and subventricular (SVZ) zones of the dorsal pallium. Newborn Indeed, the reeler mice and the mutant null mice for Dab1 or postmitotic neurons migrate out from their birth sites in the basal for both ApoER2 and Vldlr show similar positioning defects direction to form the cortical plate (CP), the cerebral cortex (Cooper, 2008; Rice and Curran, 2001; Tissir and Goffinet, anlage, where they end forming highly organized connections 2003). However, despite numerous proposals (quite often within the cortex or with subcortical targets. The mechanisms incompatible), a coherent model of reelin action to position regulating NPCs proliferation also regulate total numbers of neurons does not yet exist (Luque, 2007). Reelin signaling neurons produced at specific developmental periods and destined decoding is often assumed to occur exclusively in neurons but it to a specific neocortical layer (Caviness et al., 2009). The is even unclear whether neuronal migration per se is influenced mechanisms underlying the tight spatiotemporal link between by reelin (Mayer et al., 2006). Cells in the VZ, where primary neurogenesis and neuronal migration remain, however, less well NPCs reside, are indeed competent to respond to exogenous understood (Ge et al., 2006; Nguyen et al., 2006; Pawlisz et al., reelin by phosphorylating Dab1 and the reelin pathway is 2008). normally activated in these cells at the preplate stage of The gene mutated in reeler mouse encodes reelin, a development. Likewise, ectopic expression of reelin in the VZ glycoprotein whose function is required for laminar positioning of reeler mice promoted migration of a subset of neurons past the of cortical neurons. Reelin, synthesized and secreted by Cajal- subplate so rescuing preplate splitting during CP formation. Retzius cells in the marginal zone, is thought to deliver a signal to Nevertheless, reelin in the VZ was found not enough to establish young migrating projection neurons instructing them to reach correct cortical lamination (Magdaleno et al., 2002). Moreover, their right position. It is well established that reelin binding to reelin receptors are much more abundant in the reeler CP, Apolipoprotein E Receptor 2 (ApoER2) or Very Low Density suggesting that reelin induces their downregulation and that ApoER2 and neuronal clearance 1259

downregulation of reelin receptors begins at early/premigratory into the brain lateral ventricle, whereas on another two litters, the same amount of stages, well before neurons reach the CP (Uchida et al., 2009). EGFP plasmid was co-electroporated with either 1 mg/ml of a siRNA targeting the transcriptional product of the ApoER2 gene (AMBION s69322, validated siRNA) in More recently, reelin (cooperating with Notch signaling) has order to knock-down the expression of ApoER2, or the same concentration of a been shown both required and sufficient to determine the rate of plasmid encoding ApoER2 proteins (pMSCVpuro-mmApoER2, a kind gift from neocortical neurogenesis (Lakoma´ et al., 2011). Such interaction J. Nimpf; Hibi et al., 2009) to induce ApoER2 overexpression. For ApoER2 is also required for neuronal migration and positioning overepression experiments the position of 599 EGFP+ cells was analyzed, while 2412 EGFP+ cells were analyzed for ApoER2 siRNA experiments. Quantification of (Hashimoto-Torii et al., 2008). Thus, the early function of Dab1 signal intensity in 1332 electroporated cells was done with the NIH software reelin in the proliferative compartment might underlie its post- ImageJ. SPSS was used to perform Student t test for statistical significance.and proliferative requirement raising the possibility that acts coupling GraphPad Prism to build up the graphs. The values represent means 6 standard errors. Further methodological details and specifications are available upon request. neurogenesis and neuronal migration, perhaps akin to a permissive rather than instructive signal for cellular migration. Results and Discussion We address this question by investigating the significance of Localization of functional ApoER2 in NPCs reelin receptors regulation during mid/late neocortical development. An Alkaline Phosphatase (AP)-fusion probe of the receptor- We show that a reelin-dependent ApoER2 downregulation binding region of reelin (repeats 3 to 6, RR36) (Uchida et al., mechanism uncouples newborn neurons from NPCs, thereby 2009) was employed to assess the localization of functional reelin enabling neurons to migrate. receptors (i.e., those in the plasma membrane as mature forms) during the neocortical neurogenic phase. AP-RR36 in situ Materials and Methods binding was performed using AP fluorescent substrates Mice Heterozygous reeler mice were purchased from Jackson Laboratory (Bar Harbor, (Fig. 1A,B) or followed by double immunofluorescence ME). The nestin-reelin transgenic mice were the generous gift from T. Curran labeling of AP and several NPCs markers (Fig. 1A,C–N). (Magdaleno et al., 2002). The day of vaginal plug appearance was considered to be Radial glial cells, the primary NPCs in the VZ, were detected embryonic day 0 (E0) and genotyping performed as previously described (Lakoma´ as cell expressing Brain Lipid Binding Protein [BLBP] (Anthony et al., 2011). Animals were handled according to protocols approved by the European Union and NIH guidelines, the Nagoya University and the Instituto de et al., 2004). By embryonic day (E) 15 radial glial cell bodies in Neurociencias Animal Care and Use Committees. the basal-most part of the VZ and processes spanning the CP expressed functional reelin receptors (Fig. 1C–H). Tbr2 AP-RR36 in situ staining and densitometric analysis immunostaining was used to examine secondary NPCs in the An Alkaline Phosphatase (AP)-fusion probe of the receptor-binding region of SVZ, so called intermediate progenitor cells (IPCs) (Kriegstein reelin (repeats 3 to 6, AP-RR36) was used to detect functional reelin receptors during mid/late cortical development. Validation of the probe, and standard binding and staining procedures were previously described (Uchida et al., 2009). Here, we first avoid endogenous AP inactivation steps and conventional substrate reagents. Then, AP-RR36 in situ binding was detected using AP fluorescent substrates or, after careful fixation of free-floating slices, using anti-AP antibodies

Biology Open in combination with cell markers. A semi-quantifying densitometric analysis of standard AP-RR36 signal was performed using the Quantity One Software on 3 equal 1280 arbitrary square units per section randomly distributed through the CP region above (+/rl and +/rl ne-ree), below (rl/rl) or both above and below (rl/rl ne- ree) the estimated position of the subplate.

Immunohistochemistry and data analysis Immunostaining was performed as previously described (Lakoma´ et al., 2011). Primary and secondary antibodies were acquired and diluted as indicated: rabbit anti-Dab1 (B3, 1:500, gift from B. Howell), rabbit anti-Tbr1 (1:1000, Chemicon), rabbit anti-Cux1 (1:50, Santa Cruz), rabbit anti-Tbr2 (1:2000, gift from. R. Hevner), rabitt anti-BLBP (1:1000, Chemicon), Mouse anti-nestin (1:10 DSHB), rat anti- BrdU (crossreaction with CldU, 1:100, Serotec), mouse anti-BrdU (crossreaction with IdU, 1:100, BD), mouse anti-human Alkaline Phosphatase (1:250, Abcam), guinea pig anti-Vglut1 (1:10000, Chemicon), rat anti-GFP (1:500 Nacalni), Cy2- goat anti-mouse, Cy3-goat anti-rabbit (1:400, Jackson Immunoresearch), biotin- donkey anti-guinea pig (1:500, Jackson Immunoresearch) and Cy5-streptavidin (1:800, Jackson Immunoresearch). Unconjugated Fab fragments were used when anti-Tbr1 and anti-Cux1 were combined. Nuclei were counterstained with DAPI. Images were captured on a Leica TCS SP2 AOBS inverted Laser Scanning Confocal Microscope or a NIKON fluorescent microcope equiped with a confocal structured light system (Optigrid). Volocity 5.2, Image J (NIH, http://rsb.info.nih.gov/ij), and Adobe Photoshop software were used for image capturing and analysis.

Thymidine analogs double-labeling Chlorodeoxyuridine (CldU, Sigma, 41.66 mg/kg) and Iododeoxyuridine (IdU, Fluka, 58.33 mg/kg) were administered intraperitoneally in pregnant reelin heterozygous ne-reelin mice. CldU was injected on the 13th day of embryonic development and IdU 24 hours later. The litter was sacrificed at P0, genotyped and Fig. 1. Neocortical NPCs express functional ApoER2. In situ binding with further processed for immunofluorescence adding a DNA denaturating step. AP-RR36, an Alkaline Phosphatase-fusion probe of the receptor binding region of reelin, reveals the presence of functional reelin receptors, largely ApoER2, in Electroporation and data analysis the developing cortex at E15 (A,B). AP-RR36 staining is observed in several Electroporation-mediated in utero gene transfer was carried out on E14 mice BLBP+ radial glia processes spanning the cortical wall (C–E) as well as in embryos that were harvested at E18 or P0 as previously described (Tabata and subpopulations of radial glia cell bodies in the basal most VZ (F–H). Most Nakajima, 2001) on wild type (WT), heterozygous or reeler backgrounds. On an Tbr2+ IPCs in the SVZ (I–K) and some BrdU+ cycling progenitors (L–N) stain entire litter of animals 90 ng/ml of a plasmid encoding EGFP protein was injected also with AP-RR36. Scale bar: 200 mminB;150mm in C–E; 50 mm in F–N. ApoER2 and neuronal clearance 1260

et al., 2006). IPCs expressed high levels of functional reelin receptors (Fig. 1I–K). A short BromodeoxyUridine (BrdU, 50 mg/kg) pulse followed by BrdU immunolabeling was used to detect S-phase cycling progenitors. BrdU+ profiles in the basal most part of the VZ, the VZ/SVZ interface and the SVZ also expressed functional reelin receptors (Fig. 1L–N). By E17 AP- RR36 staining was still observed in NPCs expressing the generic NPCs marker nestin. Quite often a weaker band of receptors expression was observed within 20–30 mm from the ventricular lumen (supplementary material Fig. S1). In agreement with previous detections of antigenic determinants or mRNAs (Hartfuss et al., 2003; Kawaguchi et al., 2008; Keilani and Sugaya, 2008; Luque et al., 2003; Magdaleno et al., 2002), the expression of functional reelin receptors in both radial glia and IPCs indicates that NPCs are competent to receive reelin during the neurogenic phase of neocortical development. Consistent with this notion a previous study has demonstrated that reelin signaling regulates the temporal specification of NPCs (Lakoma´ et al., 2011). Most of the AP-RR36 signal was abolished in the ApoER2-deficient cortex while it remained virtually the same in the Vldlr-deficient one (Uchida et al., 2009; data not shown). Indeed, severe abnormalities in cortical neuronal positioning (layers II to VI) are present in the absence of ApoER2 but not of Vldlr (Benhayon et al., 2003; Hack et al., 2007). Thus, during mid/late development neocortical NPCs express mainly, if not exclusively, ApoER2. Fig. 2. ApoER2 loading determines neuronal migratory behavior. Overexpression of ApoER2 inhibits neuronal migration (A–D). P0 brains that had been transfected at E14 with an EGFP plasmid (control) (A) or with ApoER2 overexpression inhibits neuronal migration truncated (B) or full-length (C) ApoER2 plasmids are depicted. Note the ApoER2 is much more abundant in the reeler CP as compared to alignment of a layer of neurons beneath the marginal zone in control cortex wild type, suggesting that reelin induces its downregulation (A). Fewer neurons are aligned and many of them appear halted in their radial migratory trajectories in the brains transfected with ApoER2 plasmids (Uchida et al., 2009). To investigate the significance of ApoER2 (B,C). ApoER2 knock-down rescues neuronal migration and Dab1 levels in regulation, WT cortices were electroporated at E14 with either reeler (E–O). E18 cortices that had been transfected at E14 either with an Biology Open enhanced Green Fluorescent Protein (GFP, 4 embryos), with a EGFP plasmid (control) or with an EGFP plasmid plus an RNAi plasmid for full-length ApoER2 plasmid (ApoER2-full-GFP, 4 embryos) or a ApoER2 (siApoER2) are depicted. A band of aligned neurons is visible beneath plasmid with a truncated form of the receptor lacking the the marginal zone in EGFP-electroporated heterozygous cortices (E). In EGFP- electroporated reeler cortices neurons do not form an apparent band and cytoplasmic domain (ApoER2-DCD-GFP, 2 embryos) and appears confined at the bottom of the cortical wall (F). Partial rescue of examined the brains at postnatal day (P) 0. The alignment of a neuronal migration is apparent in EGFP+siApoER2-electroporated reeler layer of cells beneath the marginal zone was apparent through cortices (G). Dab1 levels are reduced in reeler cortical neurons after this temporal window in the GFP-electroporated cortices EGFP+siApoER2 electroporation (I–O). Scale bar: 150 mm in A–C,E–G; (Fig. 2A). In contrast, in the ApoER2-DCD-GFP-electroporated 15 mm in E–K,L–N. cortices fewer cells appeared aligned beneath the marginal zone and many of them were down dispersed through the CP (Fig. 2B). More dramatically, in the ApoER2-full-GFP- the probability of cell migration inhibition. This might also electroporated cortex, most cells appeared dispersed through encompass a mechanism as to why NPCs do not migrate out of the CP, the intermediate and the SVZ with very little, if any, cells the VZ/SVZ. Interestingly, by longer harvesting times (P3) many positioned just beneath the marginal zone (Fig. 2C). A cells misexpressing ApoER2-DCD-GFP showed more branched quantification of this effect is shown in Fig. 2D. The overall and thicker processes (supplementary material Fig. S2). In weaker and heterogeneous effect of ApoER2-DCD when agreement, neutralization of reelin at early postnatal stages compared with that of ApoER2-full-overexpression supports increases dendritic complexity of cortical pyramidal neurons that plasma membrane ApoER2 overloading underlies by itself involving a signal transduction pathway independent of the the migratory reeler cortical phenotype. Conceivably, our canonical reelin receptors (Chameau et al., 2009). truncated ApoER2 form competes with endogenous receptors for reelin but does not relay the intracellular signal, thus way ApoER2 knock-down rescues neuronal migration and Dab1 behaving like a dominant negative molecule (Jossin and Cooper, levels in reeler mice 2011). Then again, our full ApoER2 form might equally compete As ApoER2 overloading indeed inhibits neuronal migration in with endogenous receptors for reelin while efficiently relaying wild type cortices, ApoER2 downloading could somehow the intracellular signal. Thus, whether migration is halted in a enhance migration. However, this can hardly be tested in wild given cell would depend on the particular reelin/ApoER2 types (i.e., in the presence of endogenous reelin) when reelin stoichiometry of its plasma membrane; assuming that reelin induces ApoER2 downregulation. In fact, knocking-down of levels must be limiting, the more full receptors remain without either ApoER2 or Vldlr in wild type cortices was shown to have access to reelin and hence not being downregulated, the higher modest or no effect at all on neuronal positioning (Kubo et al., ApoER2 and neuronal clearance 1261

2010). Admittedly counterintuitive, we decide instead to knock- signaling could influence migration by regulating the cell down ApoER2 in reeler cortices. Three reelin heterozygous and 2 competence to respond to a second pathway. ApoER2 are indeed reeler cortices were electroporated at E14 with an EGFP plasmid. quite promiscuous receptors, with trombospondin-1, f-spondin, Three additional reeler cortices were electroporated with EGFP coagulation factor XI or protein C (Blake et al., 2008; Hoe et al., plasmid and an RNAi plasmid for ApoER2 (siApoER2). All 2005; May et al., 2005; White-Adams et al., 2009; Yang et al., brains were examined at E18. Again, like in WT cortices, in the 2009) among its known ligands, making conceivable the EGFP-electroporated heterozygous it was apparent the alignment existence of an inhibitory signal on cell migration that might of a layer of cells beneath the marginal zone (Fig. 2E). In be precluded upon reelin-induced ApoER2 downregulation. A contrast, in the EGFP-electroporated reeler cortices cells did not facilitated activation of ephrin Bs signaling could equally be form a discernible layer and appeared confined in the lower half involved. In fact, ephrin Bs have been recently identified as reelin of the cortical wall (Fig. 2F). However, in the EGFP+siApoER2- co-receptors as they bind directly to reelin and ApoER2/Vldlr, electroporated reeler cortices cells appeared more dispersed and resulting in Dab1 phosphorylation. Significantly, the reeler positioned in a roughly inverted pattern as compared with those cortical migratory phenotype was shown to be rescued by simple of EGFP-electroporated reeler cortices. A percentage of them activation of ephrin B signaling, i.e., in the absence of appeared beneath the marginal zone at comparable positions to endogenous reelin (Sentu¨rk et al., 2011). those of EGFP-electroporated heterozygous cortices (Fig. 2G). A quantification of these effects is shown in Fig. 2H. Disruption of Partial rescue of lamination along with ApoER2 and Dab1 levels reelin signaling by genetic ablation of reelin, ApoER2, VLDLR, by expression of reelin in NPCs of reeler mice or Fyn and Src lead to the accumulation of Dab1 protein (Arnaud The enriched expression of functional ApoER2 in NPCs and the et al., 2003; Bock and Herz, 2003; Kuo et al., 2005; Rice et al., evidence that ApoER2 loading determines the cellular migratory 1998; Sheldon et al., 1997; Trommsdorff et al., 1999), indicating behavior also predict an action of reelin in the VZ/SVZ resulting that reelin limits its action in responsive cells by reducing Dab1 in neuronal ApoER2 downregulation and positioning. To test this levels. We found that in reeler cortices the EGFP+siApoER2- prediction we took advantage of the ne-reelin mice that electroporated neurons showed lower levels of Dab1 than the ectopically express reelin in NPCs under the control of a nestin EGFP-electroporated neurons (Fig. 2I–O), raising the possibility driver (Magdaleno et al., 2002). We performed immunolabeling that ApoER2 knock-down recapitulates reelin function in the at P0 combining the use of the subplate marker VGlut1 (Ina et al., absence of endogenous reelin. Furthermore, the reduction of 2007) and the cortical layer markers Tbr1 – for layer VI and ApoER2 genetic dosage also ameliorates neuronal positioning in preplate derivatives – (Bulfone et al., 1995) and Cux1 – for layers II/III/IV (Nieto et al., 2004). We confirmed that in the WT reeler cortex (supplementary material Fig. S3). It implies that background, ectopic reelin did not alter cortical layering newborn neurons that have not activated the canonic reelin (Fig. 3A,B,E). However, against a previous notion, in the pathway retain their migratory potential suggesting that reelin reeler background ectopic reelin did partly rescue cortical Biology Open lamination, specifically in the cell population positioned above, but not that one below, an ectopic subplate (Fig. 3C,D,F). Properly positioned upper and lower layer cells contributed to the rescued CP region (i.e. above the subplate) with an approximate 15% and 70% of the total Cux1+ and Tbr1+ cell population, respectively. Consistent with previous observations (Feng and Cooper, 2009; Nieto et al., 2004), Cux1+ cells were virtually absent in the upper third of the conventional (unrescued) reeler CP (not shown). To corroborate these results we sequentially injected pregnant dams at E13 and E14 with the nucleotide analogues CldU and IdU, respectively, and harvested 4 pup brains at P0 for immunolabeling. We found that the inside-out pattern of cell positioning was partially rescued in reeler ne- reelin cortices. Indeed, later born cells (IdU+) pass earlier born cells (CldU+) over, but not beneath, the ectopic subplate (Fig. 3G,H). Thus, while the role of reelin in promoting splitting of the preplate does not seems entirely distinct from its effect on positioning, the failure of some cells to respond to reelin appears related to their migratory distance and hence to their birthdates. Interestingly, the neocortical (and cerebellar) Fig. 3. Partial rescue of inside-out patterning in reeler by ectopic reelin in phenotype of reeler ne-reelin is reminiscent of dab1cd/cd mutants, NPCs. Immunohistochemical detection at P0 of Tbr1, Cux1 and VGlut1 on lacking one out of two pair of Dab1 tyrosine phosphorylon sites adjacent sections of ne-reelin (A,B) and reeler ne-reelin (C,D) cortices. In the WT background, the expression of the ne-reelin transgene does not alter (Feng and Cooper, 2009; Morimura and Ogawa, 2009). This cortical lamination. In the reeler background it does rescue cortical layering in suggests that ne-reelin might support the kinase switch function the cell population above, but not that below, the ectopic subplate. Use of Fab of Dab1, but perhaps not sufficiently its scaffold function blocking fragments confirmed these results allowing the combination of required for the migration of upper layers neurons. By means of antibodies on the same section (E,F). Sequential injection of the nucleotide analogues CldU and IddU by E13 and E14, respectively, further demonstrate at in situ AP-RR36 staining we then evaluated the expression of P0 a rescue of the inside-out lamination pattern above the ectopic ne-reelin functional ApoER2 in ne-reelin cortices. We confirmed that at reeler subplate (G,H). Scale bar: 200 mm. E17 ApoER2 was much more abundant in the reeler than in the ApoER2 and neuronal clearance 1262

WT CP (Fig. 4A,B). In the presence of the endogenous protein properties of the neurons generated. Together, our results provide (WT or heterozygous backgrounds) ectopic reelin did not alter foundation for the highly orchestrated clearance of neocortical ApoER2 expression. In contrast, in the reeler background, projection neurons from their birth site, indicating that a reelin- ectopic reelin induced a dramatic decrease of ApoER2 above the dependent ApoER2 downregulation mechanism uncouples new- ectopic supblate (Fig. 4C,D). Comparable results were obtained born neurons from NPCs, thereby enabling neurons to migrate at P0 (Fig. 4A9–D9). A semi-quantification of these effects is (Fig. 4G). Further elucidation of the underlying molecular shown in Fig. 4E,F. Moreover, normalized Dab1 levels were mechanisms of ApoER2 regulation and mediated signaling observed at P0 in the rescued CP region of reeler ne-reelin mice (including reelin and ligands other than reelin as well as putative (supplementary material Fig. S4), further supporting that at least co-receptors) should reveal novel concepts and patterns that in part, the function of reelin in the proliferative compartment provide a clear link between NPCs proliferation/neuronal fate underlies its post-proliferative requirement for neuronal migra- and neuronal migration. It might become increasingly evident tion and positioning. The recent claim that deletion of Dab1 in that neurogenesis and neuronal positioning are less separately migrating neurons alone phenocopies reeler lamination defects encoded than it has hitherto been thought. (Franco et al., 2011) seems apparently incompatible with our proposal. However, since the NEX promoter element used by Acknowledgements Franco et al. (Franco et al., 2011) can drive expression in IPCs We thank Drs S. Magdaleno and T. Curran for ne-reelin mice, Drs B. (Goebbels et al., 2006), it cannot be ruled out that Dab1 deletion Howell and R. Hevner for Dab1 and Tbr2 antibodies, Dr J. Nimpf for ApoER2 plasmids, M. Masaoka for outstanding technical help with in IPCs might have contributed to effects on the migratory surgery and electroporation, and Drs L. Garcıá-Alonso and J. Lakoma´ for support and discussions. F.J.P.-M. held graduate fellowships from the Universidad Miguel Hernańdez/FundaciońMe´dica Mutua Madrilenã (FMMM) and the Japan Society for the Promotion of Science (Global COE Program). This work was supported in part by grants from Japanese Grant-in-Aids for Scientific Research on Innovative Area (Neural Diversity and Neocortical Organization), and for Scientific Research (B) [to M.H.]; KAKENHI 20021016 and 22111006 [to T.M.]; the Spanish Ministry of Science and Innovation SAF2004-07685 and the FMMM [to J.M.L.].

Competing Interests The authors have no competing interests to declare.

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